Drain down and re-feed of microcarrier bioreactor

ABSTRACT

Disclosed is a method of increasing product yield per culture in a population of bound product-secreting cells in a bioreactor, the method comprising: semi-harvesting product by removing a volume of the culture medium with a first-secreted product concentration; re-feeding the bound population of product-secreting cells by adding an amount of a fresh culture medium sufficient to increase the volume of the culture medium to approximately the original volume of the culture medium; agitating the culture medium under sufficient conditions and for a sufficient time period to allow the bound population of product-secreting cells to grow and to release a second-secreted product concentration into the culture medium; and harvesting product.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2010/043013, which designated the United States and was filed onJul. 23, 2010, published in English, which claims priority to U.S.Provisional Patent Application No. 61/228,026, filed on Jul. 23, 2009.The entire teachings of the above applications are incorporated hereinby reference.

BACKGROUND

Cell culturing is an essential step in manufacturing biological productssuch as, for example, nucleic acids, viruses for use in vaccines,antibodies, and proteins, for example, interferons. Anchorage-dependentcells, such as certain animal cells, need to attach to a surface inorder to grow and divide.

For large-scale cell culturing, microcarriers provide the large surfacearea needed for growing anchorage-dependent cells. Van Wezel, in 1967,described the use of microcarriers, small beads or particlesapproximately 0.2 mm in diameter, for growing such cells. Using gentleagitation, the microcarriers to which the cells will attach aresuspended in a liquid culture medium within a bioreactor.

The process may begin with the addition of cells (the inoculum) to theliquid culture medium in which the microcarriers are suspended. Theculture medium contains the nutrients essential for metabolism andgrowth of the cells. Conditions of temperature, pH, and oxygenconcentration are controlled to promote cell growth and division inorder to increase cell density and confluence.

Continuous or Perfusion Mode: In a continuous or perfusion mode,nutrients are continuously added to the system, and product is harvestedthroughout the culture period. With the continuous mode, the on-goingdifficulty in obtaining sufficiently high product titers is wellrecognized. In addition to the low titer issue, there is a need toconcentrate product of the continuous mode. These problems have a directimpact on production time and cost, and make the continuous mode lessfeasible at least for vaccine production.

Batch Mode: In a batch mode, all nutrients are added at the beginningand products are not removed until the end of the batch. Waste productsaccumulate during the run, and nutrients are used up, making the batchprocess inefficient for many applications.

Fed-Batch Mode: A fed-batch mode is similar to the batch mode in thatproducts are removed only at the end of the run, but differs in thatnutrients are added at multiple intervals during the process. Mostvirus-producing, microcarrier cultures are carried out, post infection,in a fed-batch process. In the fed-batch mode, there is also an increasein waste products and other contaminants, such as host cell protein andhost cell DNA, and dead cells falling off of the microcarriers.

Thus, there remains an on-going need for a new process that increasesproduct titer while eliminating the problems inherent in the batch modeand the fed-batch mode of cell culturing. Moreover, techniques that canincrease the yield, production efficiency or speed of harvesting viralproducts for vaccines, in particular, would also satisfy an on-goingneed and permit the health care system to respond more rapidly to newviral outbreaks.

SUMMARY OF THE DISCLOSURE

In contrast to prior art methods for producing virus in mirocarriercultures, the inventors of the present subject matter have nowdiscovered a new method of harvest and re-feed for culturing, postinfection, virus-producing cells on a scaffold such as a microcarrier, amethod that significantly increases product titer while reducing theconcentration of contaminants in the culture. The disclosed“semi-harvest and re-feed method, also referred to herein as the “draindown and re-feed method,” and the “harvest and re-feed method,” is alsoapplicable to the culturing of other product-secreting cells on ascaffold, for example a microcarrier, to harvest diverse products, suchas antibodies, proteins, hormones, peptides and growth factors. Anexample of a protein product is an interferon. The invention, interalia, includes the following, alone or in combination.

In one aspect, the present invention relates to a method of increasingproduct yield per culture in a population of product-secreting cellsbound to a scaffold at least partially immersed in an original volume ofa culture medium in a bioreactor, the method comprising: semi-harvestingproduct by removing from the bioreactor a first portion of the originalvolume of the culture medium with a first-secreted product concentrationfrom the bioreactor while leaving the scaffold with the bound populationof product-secreting cells in the bioreactor; re-feeding the boundpopulation of product-secreting cells by adding to the bioreactor anamount of a fresh culture medium sufficient to increase the volume ofthe culture medium in the bioreactor to approximately the originalvolume of the culture medium; agitating the culture medium in thebioreactor under sufficient conditions and for a sufficient time periodto allow the bound population of product-secreting cells to grow and torelease a second-secreted product concentration into the culture medium;and harvesting product by removing from the bioreactor at least aportion of the culture medium with the second-secreted productconcentration from the bioreactor while leaving the scaffold with thebound population of product-secreting cells in the bioreactor. Thescaffold can optionally be a microcarrier, e.g., microcarrier beads.This process can continue as long as the cells remain viable to yieldthird product concentrations, fourth product concentrations, etc.

In another aspect, the present invention relates to a method forincreasing virus yield per culture in a population of virus-infectedcells bound to a microcarrier suspended in an original volume of aculture medium in a bioreactor, the method comprising: semi-harvestingvirus by removing from the bioreactor a first portion of the originalvolume of the culture medium with a first-shed virus while leaving themicrocarrier with the bound population of virus-infected cells and aremaining volume of the culture medium in the bioreactor; re-feeding thebound population of virus-infected cells by adding to the bioreactor anamount of a fresh culture medium sufficient to increase the remainingvolume of the culture medium in the bioreactor to approximately theoriginal volume of the culture medium; agitating the culture medium andthe microcarrier with the bound population of virus-infected cells undersufficient conditions and for a sufficient time period to allow thevirus to continue to infect the bound population of virus-infected cellsand to allow the bound population of virus-infected cells to release asecond-shed virus into the culture medium; and harvesting virus byremoving from the bioreactor at least a portion of the culture mediumwith the second-shed virus from the bioreactor while leaving themicrocarrier with the bound population of virus-infected cells in thebioreactor. Again, this process can be further repeated to yieldadditional harvests of virus for as long as the virus-infected cellsremain viable.

Another embodiment of the invention is a method of increasing virusyield per culture in cells growing on a conditioned microcarrier in abioreactor, the method comprising: transferring a plurality of seedcells into the bioreactor containing the conditioned microcarrier and anoriginal volume of a culture medium, to form in the bioreactor a mixturecomprising the seed cells, the culture medium, and the microcarrier;agitating the mixture in the bioreactor at a sufficient rate ofagitation and for a sufficient time to allow the seed cells to bind tothe microcarrier; cultivating the seed cells bound to the microcarrierunder sufficient conditions and for a sufficient time period for theseed cells to form a bound cell population that is from about 35 percentto about 95 percent confluent on the microcarrier; removing from thebioreactor from about 30 percent to about 88 percent of the originalvolume of the culture medium in the bioreactor, while leaving themicrocarrier with the bound cell population in the bioreactor, to form afirst reduced volume of culture medium; infecting the bound cellpopulation with a virus; allowing the virus to adsorb to the bound cellpopulation and to infect the bound cell population; re-feeding the boundcell population including the virus adsorbed thereto by adding to thebioreactor a first amount of a fresh culture medium sufficient toincrease the first reduced volume of the culture medium in thebioreactor to approximately the original volume of the culture medium;agitating the culture medium and the microcarrier with the bound cellpopulation under sufficient conditions and for a sufficient time periodto allow the virus to infect the bound cell population and to allow theinfected, bound cell population to release a first-shed virus into theculture medium; semi-harvesting virus by removing from the bioreactor aportion of the culture medium with the first-shed virus, the portion ofthe culture medium removed equal to from about 50 percent to about 90percent of the original volume of the culture medium, while leaving themicrocarrier with the infected, bound cell population, and a secondreduced volume of the culture medium in the bioreactor; re-feeding thebound cell population by adding to the bioreactor a second amount of afresh culture medium sufficient to increase the second reduced volume ofthe culture medium in the bioreactor to approximately the originalvolume of the culture medium; agitating the culture medium and themicrocarrier with the bound cell population in the bioreactor undersufficient conditions and for a sufficient time period to allow thevirus to continue to replicate in the bound cell population and to allowthe infected, bound cell population to release a second-shed virus intothe culture medium in the bioreactor; and harvesting virus by removingfrom the bioreactor at least a portion of the culture medium with thesecond-shed virus while leaving the microcarrier with the infected,bound cell population in the bioreactor.

The invention also relates to a method of increasing virus yield perculture in cells growing on a conditioned microcarrier in a bioreactor,the method comprising: transferring a plurality of seed cells into thebioreactor containing a microcarrier and an original volume of a culturemedium; cultivating the seed cells to bind to the microcarrier and forma bound cell population that is from about 35 percent to about 95percent confluent on the microcarrier; removing from the bioreactor fromabout 30 percent to about 88 percent of the original volume of theculture medium in the bioreactor, while leaving the microcarrier withthe bound cell population in the bioreactor, to form a first reducedvolume of culture medium; infecting the bound cell population with avirus, which can optionally be a flavivirus; adding fresh culture mediumto the bioreactor to maintain the bound cell population; culturing thebound cell population for a sufficient time period to allow theinfected, bound cell population to release a shed virus concentrationinto the culture medium in the bioreactor; and harvesting virus byremoving from the bioreactor at least a portion of the culture mediumwith shed virus therein.

In another aspect of the invention, methods of increasing virus yieldper culture in cells growing in a bioreactor are disclosed, comprisingthe steps of: transferring a plurality of seed cells into the bioreactorcontaining a support substrate, such as conditioned microcarriers, andan original volume of a culture medium, to form in the bioreactor amixture comprising the seed cells, the culture medium, and thesubstrate; and cultivating the seed cells bound to the substrate undersufficient conditions and for a sufficient time period for the seedcells to form a bound cell population that is optionally from about 35percent to about 95 percent confluent on the substrate.

Once a bound cell population is formed, from about 30 percent to about88 percent of the original volume of the culture medium in thebioreactor is removed, while leaving the substrate with the bound cellpopulation in the bioreactor, to form a first reduced volume of culturemedium; infecting the bound cell population with a virus; allowing thevirus to adsorb to the bound cell population and to infect the boundcell population. Fresh culture medium can then be added to thebioreactor and the bound cell population is cultured under sufficientconditions and for a sufficient time period to allow the virus to infectthe bound cell population and to allow the infected, bound cellpopulation to release a virus into the culture medium. The virus canthen be harvested by the above-described drain down and re-feed methodor any other known harvesting techniques.

DETAILED DESCRIPTION

A description of preferred embodiments of the invention follows. It willbe understood that the particular embodiments of the invention are shownby way of illustration and not as limitations of the invention. At theoutset, the invention is described in its broadest overall aspects, witha more detailed description following. The features and other details ofthe compositions and methods of the invention will be further pointedout in the claims.

The present disclosure relates to the production of cells onmicrocarriers or other structures for cell attachment, the microcarrierssuspended in bioreactors, including, for example, spinner flasks, benchtop bioreactors, and larger non-disposable and disposable bioreactors.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of these words mean “includingbut not limited to”, and they are not intended to (and do not) excludeother moieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims and abstract), and/or all of thesteps of any method or process so disclosed, may be combined in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive. The invention is not restricted tothe details of any foregoing embodiments. The invention extends to anynovel one, or any novel combination, of the features disclosed in thisspecification (including any accompanying claims and abstract), or toany novel one, or any novel combination, of the steps of any method orprocess so disclosed.

Typically, in the traditional fed-batch process of growingvirally-infected cells, as the culturing of cells progresses, a plateauin viral titer is reached, accompanied by an increase in concentrationsof the contaminating host cell protein (HCP), the host cell DNA, andwaste products. When the plateau in viral titer is reached, theproduction of virus has significantly decreased or stopped altogether.At that time, the process is discontinued; the culture medium with shedvirus is harvested; the microcarriers with the dead cells are discarded;and a new culture set up. There has been an on-going need to improvethis process in order to increase the viral titer achieved within agiven period of time, to speed the production of vaccines.

The inventors of the present subject matter have now discovered a newmethod of culturing anchorage-dependent cells on a scaffold, a methodthat addresses the potential problem of metabolite depletion and wasteproduct build-up and which provides increased product titer within agiven time period as compared to the amount of product titer achievedwithin the same time period using the fed-batch process. Typically,suspension cultures are maintained in serum free media.

The disclosed method provides for a supply of specific nutrients, growthfactors, lipids, amino acids, vitamins, salts, and trace metals byincreasing their relative concentration during culture to an optimallevel, and removing contaminants, thus improving not only the cellgrowth rates, but also culture density, specific productivity and/orspecific product quality and concentration.

An embodiment of the invention provides a “drain-down” or “semi-harvestand re-feed” step that significantly reduces the concentration ofmetabolic waste products and cellular byproducts such as host cellprotein and host cell DNA. Host cell protein and host cell DNA arecontaminants that interfere with production of a virus for use in avaccine, for example. The drain-down step can remove a large portion ofthe contaminated culture medium, and the re-feed step replenishes thenutrient-depleted, contaminated culture medium by adding fresh medium.

In one aspect, the invention provides for the use of a harvest orsemi-harvest and re-feed of an infected attachment culture, including,but not limited to virus-producing microcarrier cultures, in order toincrease virus titer, for example. In another aspect, the inventionprovides for increasing product yield per culture in a population ofproduct-secreting cells bound to a scaffold.

Disclosed herein is a method for sterilely removing a volume of theconditioned culture medium, for example, the removal of aboutseventy-five percent (75%) of the culture medium, while retaining in thebioreactor the total cell population bound to a microcarrier. Thissemi-harvest is followed by replacement with an equal volume of freshculture medium. The culturing process is allowed to continue.

As the terms are used, herein, “semi-harvest” and “harvest” havesubstantially the same meaning and are sometimes used interchangeably.“Semi-harvest” is often used to denote a step in which the culturemedium is drained down, for example, using a sieve, to collect the virusthat was shed into the culture medium during an early phase of theculturing process. “Harvest” is generally used herein to denote thefinal drain-down of the culture medium to collect the virus shed duringa later phase of the culturing process. The final harvest is typicallydone when the cytopathic effect (CPE) is from about 60 percent to about90 percent, or at about 80 percent. As the terms are used herein, a“liter” is denoted by “L” and a milliliter or cubic centimeter isdenoted by “ml”.

One embodiment of the present invention relates to a method ofincreasing product yield per culture in a population ofproduct-secreting cells bound to a scaffold immersed in a culture mediumin a bioreactor. The scaffold can optionally be a microcarrier, such asmicrocarrier beads. The method includes: semi-harvesting the product byremoving a first portion of the culture medium which includes afirst-secreted product concentration, while leaving the scaffold withthe bound population of product-secreting cells behind in thebioreactor; then re-feeding the bound population of product-secretingcells by adding fresh culture medium in an amount sufficient to increasethe volume of the culture medium to approximately the original volume ofthe culture medium. Next, the culture medium in the bioreactor isagitated under sufficient conditions and for a sufficient time period toallow the bound population of product-secreting cells to grow and torelease a second-secreted product concentration into the culture medium.Finally, when product titers have approximately peaked, the product isharvested by removing from the bioreactor at least a portion of theculture medium with the second-secreted product concentration from thebioreactor while leaving the scaffold with the bound population ofproduct-secreting cells in the bioreactor.

Another embodiment of the present invention is a method for increasingvirus yield per culture in a population of virus-infected cells bound toa microcarrier suspended in an original volume of a culture medium in abioreactor. This embodiment of the method includes: semi-harvestingvirus by removing a first portion of the original volume of the culturemedium with a first-shed virus while leaving the microcarrier with thebound population of virus-infected cells and a remaining volume of theculture medium in the bioreactor; then re-feeding the bound populationof virus-infected cells by adding to the bioreactor an amount of a freshculture medium sufficient to increase the remaining volume of theculture medium in the bioreactor to approximately the original volume ofthe culture medium; then agitating the culture medium and themicrocarrier with the bound population of virus-infected cells undersufficient conditions and for a sufficient time period to allow thevirus to continue to infect the bound population of virus-infected cellsand to allow the infected, bound population of virus-infected cells torelease a second-shed virus into the culture medium. Finally, at thetime of approximately peak titers, virus is harvested by removingculture medium with the second-shed virus from the bioreactor whileleaving the microcarrier with the infected, bound population ofvirus-infected cells in the bioreactor.

In yet another embodiment is a method of increasing virus yield perculture in cells growing on a conditioned microcarrier in a bioreactor,the method including: transferring a plurality of seed cells into thebioreactor containing the conditioned microcarrier and an originalvolume of a culture medium. The bioreactor at this point contains amixture comprising the seed cells, the culture medium, and themicrocarrier. The mixture in the bioreactor is agitated at a sufficientrate and for a sufficient time to allow the seed cells to bind to themicrocarrier. The bound seed cells are cultivated under sufficientconditions and for a sufficient time period for the seed cells to form abound cell population that is from about 35 percent to about 95 percentconfluent on the microcarrier. At this time a drain-down or semi-harvestis performed, thereby removing from the bioreactor from about 30 percentto about 88 percent of the original volume of the culture medium in thebioreactor, while leaving the microcarrier with the bound cellpopulation in the bioreactor. There is now a first reduced volume ofculture medium in the bioreactor. The bound cell population is infectedwith a virus, which is allowed to adsorb to the bound cell populationand to infect the bound cell population. Then the bound cell populationincluding the virus adsorbed thereto is re-fed with fresh medium, byadding first amount of a fresh culture medium sufficient to increase thefirst reduced volume of the culture medium in the bioreactor toapproximately the original volume of the culture medium. Again theculture medium and the microcarrier with the bound cell population isagitated under sufficient conditions and for a sufficient time period toallow the virus to infect the bound cell population and to allow theinfected, bound cell population to release a first-shed virus into theculture medium.

The virus is then semi-harvested by removing from the bioreactor aportion of the culture medium with the first-shed virus, the portion ofthe culture medium removed equal to from about 50 percent to about 90percent of the original volume of the culture medium, while leaving themicrocarrier with the infected, bound cell population, and a secondreduced volume of the culture medium in the bioreactor. Then the boundcell population is re-fed by adding a second amount of a fresh culturemedium sufficient to increase the second reduced volume of the culturemedium in the bioreactor to approximately the original volume of theculture medium. Agitating the culture medium and the microcarrier withthe bound cell population is continued under sufficient conditions andfor a sufficient time period to allow the virus to continue to infectthe bound cell population and to allow the infected, bound cellpopulation to release a second-shed virus into the culture medium in thebioreactor. Finally, the virus is harvested by removing the culturemedium with the second-shed virus while leaving the microcarrier withthe infected, bound cell population in the bioreactor.

In the foregoing example, the semi-harvesting of the virus may compriseremoving about 75 percent of the culture medium with the first-shedvirus from the bioreactor.

In any of the above-described examples, the bioreactor may be, forexample, a disposable or a non-disposable bioreactor having a volume offrom about 25 liters to about 200 liters. In another embodiment,bioreactor is chosen from a bench-top bioreactor and a spinner flask.

In some embodiments, the cells cultured are VERO cells.

The term “virus” as used herein is intended to cover not only completeinfectious viral particles but also any other secreted products that canbe used to immunize a subject, including for example, attenuatedviruses, genetically engineering viruses that are defective, e.g., intheir envelope, nucleocapsid, or genome, viral fragments and any otherviral derivatives suitable for use in vaccines or screening assays. Insome embodiments, the virus is a Flavivirus. Examples of Flavivirusesinclude: St. Louis encephalitis, Japanese encephalitis, tick-borneencephalitis viruses, dengue virus, Kyasanur Forest disease virus, andYellow Fever virus.

EXEMPLIFICATION Example 1 Benchtop Bioreactor Production withMicrocarriers and VERO Cells

SUMMARY: Using a 10 L New Brunswick (NBS) benchtop bioreactor operatingat 8 L culture volume with 5 g/L microcarriers, we prepared HYPERFLASK®(CORNING®, Corning N.Y.) seed cultures and the benchtop bioreactor;performed infection, semi-harvest and re-feed on the bioreactor.

Pre-Seed Preparation:

-   -   Set-up (8) HYPERFLASKS®, inoculated approximately 1:6 from (1        or 2) confluent HYPERFLASKS®, incubated at 37° C. in humidified        5% CO₂ incubator.

Benchtop Bioreactor and Microcarrier Preparation:

-   -   a. Prepared 40 grams of CYTODEX® I microcarriers (GE Healthcare        Bio-Sciences AB, Uppsala, SWEDEN) by hydrating more than 4 hours        in Phosphate buffered saline (PBS) (50 mL/g beads). Note: 1 g        beads swells to 20 mL volume.    -   b. Following hydration, aspirated the hydrating PBS and replaced        with fresh PBS.    -   c. Autoclaved beads at 123° C. for 60 min liquid cycle.    -   d. Prepared 10 liter benchtop bioreactor, that is, calibrated        probes, assembled tubing and interior harvest sieve tube.    -   e. Autoclaved bioreactor at 121° C. for 60 min liquid cycle.    -   f. Allowed to cool in BioSafety cabinet (BSC).    -   g. From microcarriers, aspirated PBS; introduced 3 L culture        medium (OPTIPROT™, glucose, salts, serum-free medium—Invitrogen,        Carlsbad, Calif.).    -   h. Mixed, allowed to settle, let stand at least 1 hour. Then        aspirated medium. Introduced fresh medium up to 2 L volume.    -   i. Sterily introduced approximately 6.8 L of culture medium into        the bioreactor.    -   j. Sterily transferred the conditioned microcarriers into the        bioreactor and allowed to mix, agitating at about 50 rpm for        approximately an hour in order to allow the reactor to        equilibrate.

Benchtop Bioreactor Seeding (21 Sep. 8):

-   -   a. Harvested cells from (8) confluent HYPERFLASKS®. The harvest        volume was approximately 1.2 L. Performed cell count.    -   b. Introduced the seed suspension into the awaiting bioreactor.        This resulted in a final initial viable cell (VC) density of        approximately 4E5VC/mL after the reactor is Quantity Sufficient        (QS) to 8 L final volume.    -   c. Agitated at 50 rpm for 1 minute in order to mix cells and        beads.    -   d. Stopped agitation for approximately 30 minutes.    -   e. Agitated at 50 rpm for 1 minute.    -   f. Stopped agitation for approximately 30 minutes.    -   g. Agitated at 50 rpm for about 5 minutes. Pulled sample for        visual examination to ensure cell attachment to the beads.    -   h. Attachment was apparent, and therefore, then cultivation and        reactor parameters were set.    -   i. Sampled daily for visual observation, metabolite analysis and        nuclei count.    -   After several days of cultivation, when beads were 70-80%        confluent, the culture was ready for infection with viral stock.

Infection Procedure:

a. Viral stock

-   -   i. Obtained attenuated Yellow Fever viral stock from −80° C.        storage. Allowed to thaw at room temperature.    -   ii. In a BSC, introduced appropriate amount of viral stock into        100 mL of serum free medium (OPTIPRO™, Invitrogen, Carlsbad,        Calif.) in transfer apparatus, amount appropriate in order to        achieve target Multiplicity Of Infection (MOI), that is, ratio        of infectious virus particles to Vero cells, equal to about        0.01.

b. Bioreactor readiness

-   -   i. Turned off agitation and other parameter controls on the        bioreactor and allowed the beads with cells to settle.    -   ii. Once settled, aseptically removed approximately 7 L of        medium via the sieve tube, avoiding the removal of beads.        (Needed to remove from about 40 percent to about 88 percent of        medium).    -   iii. Introduced the above referenced viral stock (step 4.a.i).    -   iv. Once introduced, returned the transfer apparatus to the BSC        and added 100 ml medium in order to chase viral stock in        transfer line into the reactor completely.    -   v. Turned on agitation at 30 rpm with other parameter controls        off for 1 hour.    -   vi. After 1 hour, introduced fresh medium up to the 8 L volume        and increased agitation to 50 rpm and turned on all parameter        controls to the original settings.

Drain-Down/Semi-Harvest and Re-Feed of Infected Culture (30 Sep. 08):

-   -   a. Following approx. 9 days of cultivations and monitoring,        drained down or semi-harvested the conditioned medium containing        the first-shed virus.        -   i. Turned off agitation and other parameter controls on the            bioreactor and allowed the beads/cells to settle.        -   ii. Once settled, aseptically drained down approximately 6 L            of medium via the sieve tube, avoiding the removal of beads.        -   iii. After draining down of conditioned medium with            first-shed virus, introduced fresh medium up to the 8 L            total volume and increased agitation to 50 rpm and turn on            all parameter controls to the original settings.

Final Harvest: Second-Shed Virus (4 Oct. 2008)

-   -   Following approx. 4 days of cultivations and monitoring,        harvested the conditioned medium containing the second-shed        virus.        -   i. Turned off agitation and other parameter controls on the            bioreactor and allowed the beads/cells to settle.        -   ii. Once settled, aseptically drained down approximately 90            percent of medium via the sieve tube, avoiding the removal            of beads.        -   iii. Plaque Forming assay performed. Viral titer obtained            was 7.25E+06 Plaque Forming Units (PFU) per nil. The results            are displayed in TABLE 1 below.

Example 2 Spinner Flask Production with CYTODEX® I Microcarriers andVERO Cells

Summary: Prepared (12) T-150 flasks. Seeded (2) 500 mL seed spinnerflasks (300 mL working volume) each at approximately 4E+05 VERO CELLS/ml(VC/mL) and 5 g/L microcarrier beads, respectively. (This is done toprovide a theoretical value of 18.6 cells/bead). Used each seed spinnerculture to inoculate each of (2) “receiving” spinner flask cultures, allat 4E+05 VC/mL and 5 g/L microcarriers, respectively.

1) Pre-Seed Preparation:

-   -   a. Set-up (12) T-150 flasks inoculated 1:6 from (2) confluent        T-150 flasks. Incubated at 37° C. for approximately 72 hours.

2) Seed Spinner Flask and Microcarrier Preparation:

-   -   a. Prepared 3 grams of CYTODEX® I microcarriers by hydrating >4        hours in PBS (50 mL/g beads). Note: 1 g beads swells to 20 mL        volume.    -   b. Following hydration, aspirated hydrating PBS and replaced        with fresh PBS.    -   c. Autoclaved at 123° C. for 60 min liquid cycle.    -   d. Allowed to cool in BSC. Aspirated PBS and introduced 300 mL        VP-SFM medium.    -   e. Mixed; allowed to settle, letting stand at least 1 hour. Then        aspirated medium. Introduced fresh medium up to 200 mL volume.    -   f. Aliquoted 100 mL of medium with 1.5 g of beads (should be ˜30        mL bead volume) into each of (2) 500 mL spinner flasks. Placed        in 37° C. incubator at 40 rpm and allowed to equilibrate for >1        hour.

3) Seed Spinner Inoculation:

-   -   a. Harvested cells from (12) confluent T-150 flasks. The harvest        volume was approximately 150 mL. Performed cell count.    -   b. Aliquoted volume (approximately 75 mL) of cells suspension        equal to approximately 1.2E8 total viable cells (VC) into each        of (2) seed spinners flasks (reference above 2f). This resulted        in a final initial viable cell density of approximately 4E5        VC/mL after the spinners are QS to 300 mL final spinner volume        each.    -   c. Placed in 37° C. incubator. Agitated at 40 rpm for 1 minute        in order to mix cells and beads.    -   d. Stopped agitation for approximately 30 minutes.    -   e. Agitated at 40 rpm for 1 minute.    -   f. Stopped agitation for approximately 30 minutes.    -   g. Agitated at 40 rpm. Moved flask to the hood and pulled sample        for visual examination to ensure cell attachment to the beads.    -   h. Attachment was apparent, and therefore, then each spinner        flask was brought to QS-300 mL with warm VP-SFM medium. If no        attachment, we would have repeated steps 3d-g.    -   i. Returned spinner flask to the incubator and turned on        agitation at 50 rpm.    -   j. Sampled daily for visual observation, metabolite analysis and        nuclei count.    -   k. When beads were 70-80% confluent, the culture was ready for        use in seeding next flasks.    -   l. Note: Each seed spinner flask should be sufficient to        seed (2) subsequent flasks as described below.

4) Receiving Spinner Flask and Microcarrier Preparation:

-   -   a. Prepared 6 grams of CYTODEX® I microcarriers by hydrating >4        hours in PBS (50 mL PBS/g beads). Note: 1 g beads swells to 20        mL volume. This was enough to prepare (4) 500 mL spinner flasks        at 400 mL final culture volume.    -   b. Following hydration, aspirated hydrating PBS and replaced        with fresh PBS.    -   c. Autoclaved at 123° C. for 60 min liquid cycle. Caution with        Handling.    -   d. Allowed to cool in BSC, then aspirated PBS and introduced        VP-SFM medium to a 400 mL total volume.    -   e. Mixed and allowed to settle, letting stand at least 1 hour.        Then aspirated medium. Introduced fresh medium up to 400 mL        volume.    -   f. Aliquoted 100 mL of medium with 1.5 g of beads (should be ˜30        mL bead volume) into each of the 500 mL spinner flasks. Placed        them in 37° C. incubator at 40 rpm and allowed to equilibrate        for >1 hour.

5) TrypLE™ (Invitrogen) Experimental Spinners Set-up:

-   -   a. Transferred (1) of the above referenced seed spinner flasks        (reference 3j) into the BSC.    -   b. Allowed to settle, then aspirated medium.    -   c. Introduced 100 mL PBS and gently swirled to mix.    -   d. Allowed to settle and aspirated PBS.    -   e. Introduced 40 mL TrypLE™ (Invitrogen) to each flask. Gently        swirled to mix. Transferred flask into 37° C. incubator and        agitated at 40 rpm for 10 minutes.    -   f. Transferred (2) of the above referenced “receiving” spinner        flasks (reference 4f) into the BSC.    -   g. Removed seed spinner from incubator and in BSC introduced 100        mL medium. Returned to incubator and agitated at 60 rpm for 2        min.    -   h. In BSC, swirled the seed flask to ensure uniform suspension.        Aliquoted approximately 44 mL into each of awaiting “receiving”        flasks (reference 6f).    -   i. Placed each in 37° C. incubator. Agitated at 40 rpm for 1        minute in order to mix cells and beads.    -   j. Stopped agitation for approximately 30 minutes.    -   k. Agitated at 40 rpm for 1 minute.    -   l. Stopped agitation for approximately 30 minutes.    -   m. Agitated at 40 rpm. Moved flask to the hood and pulled sample        for visual examination to ensure cell attachment to the beads.    -   n. Attachment was apparent. Then QS each spinner flask to 400 mL        with warm VP-SFM™ (Invitrogen, Carlsbad, Calif.) serum free        medium. (If not QS, we would have repeated steps 6j-m.) To QS to        400 mL final volume, this means adding approximately 256 mL        fresh medium.    -   o. Returned spinner flask to the incubator and turned on        agitation at 50 rpm.    -   p. Sampled daily for visual observation, metabolite analysis,        nuclei count and trypsin count for viability determination.

Results for Example 1 Benchtop Bioreactor Production with Microcarriersand VERO Cells

The results of the Example 1 test run of the disclosed drain-down andre-feed method, as performed in a 10 Liter bench top bioreactor areshown in the Table below. The Table shows the viral count in plaqueforming units (PFU) at Time zero at 2.50E+02, and reaching a peak of1.15E+08 at 48 hours, then decreasing to 1.01E+07 at 96 hours. The besttime to harvest would have been at the peak titer, reached at 48 hours.

In subsequent cultures utilising the disclosed method, we typicallyseeded the microcarriers at from about 4E+05 VC/ml to about 6E+05 VC/ml.We then typically infected at about 48 hours post-seed, when the cellsare about 85 percent (85%) confluent. Infection may also be done whenthe bound cell population is from about 35 percent to about 95 percentconfluent on the microcarrier. In one embodiment of the invention, atinfection, the concentration of cells is greater than or equal to about7E5 VC/ml.

We generally perform a drain-down or semi-harvest and re-feed at about72 hours post infection. The final harvest is usually carried out atabout 52 hours post re-feed. The best concentration of viable cellsseeded on the microcarriers, and the best times for infecting,drain-down, and final harvest will of course vary with the type of virusused, and can be determined without undue experimentation by those ofskill in the art.

The VERO host cell protein and DNA are also shown in the Table.

Analysis: By using the above-disclosed drain-down and re-feed method, wewere able to obtain approximately 10 percent (10%) greater viral titerin a time period of about seven (7) days than the viral titer weobtained with the traditional fed-batch process in a similar timeperiod. This was an unexpected result, but a fortunate outcome becausethe disclosed method achieves a significant savings in cost of vaccineproduction over the fed-batch method and a significant savings in time.When vaccines are needed urgently, the disclosed method can help tospeed production of the vaccines. The levels of host cell protein andDNA contaminants are also lowered when using the disclosed method ratherthan the batch fed method.

We are also performing the disclosed method in a 200 Liter bioreactorand plan to scale up to a 500 liter bioreactor.

TABLE DNA/ml by Vero cell Plaque picogreen Vero protein/ml Sample Sampleassay DNA assay assay protein by ELISA number description Expt # PFU/mlExpt # μg/mL Expt # μg/mL Y0183A T-0 T-0 Y0190A 2.50E+02 Y0199A 0.23Y0188A 18.392 (Infection) Y0183A 24 Hr Y0190A 2.50E+04 Y0199A 0.18Y0188A 14.757 Y0183A 48 Hr Y0190A 2.50E+04 Y0199A 0.26 Y0188A 25.068Y0183A 72 Hr Y0190A 5.00E+05 Y0199A 0.34 Y0188A 30.752 Y0183A 96 HrY0190A 1.01E+07 Y0199A 0.47 Y0188A 48.819 (Drain down) Y0183A (A) T-0(One Y0213A 7.25E+06 Y0206A 0.39 Y0205A 25.445 hour after Re-Feed) 30Sep. 2008 Y0183A (A) 24 Hr Y0213A 8.50E+07 Y0206A 0.3 Y0205A 34.106Y0183A (A) 48 Hr Y0213A 1.15E+08 Y0206A 0.69 Y0205A 63.131 Y0183A (A) 72Hr Y0213A 2.05E+07 Y0206A 1.2 Y0205A 95.818 Y0183A (A) 96 Hr Y0213A7.25E+06 Y0206A 1.4 Y0205A 147.463

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of increasing product yield per culture in a population ofproduct-secreting cells bound to a scaffold, which can optionally be amicrocarrier, at least partially immersed in an original volume of aculture medium in a bioreactor, the method comprising: semi-harvestingproduct by removing from the bioreactor a first portion of the originalvolume of the culture medium with a first-secreted product concentrationfrom the bioreactor while leaving the scaffold with the bound populationof product-secreting cells in the bioreactor; re-feeding the boundpopulation of product-secreting cells by adding to the bioreactor anamount of a fresh culture medium sufficient to increase the volume ofthe culture medium in the bioreactor to approximately the originalvolume of the culture medium; agitating the culture medium in thebioreactor under sufficient conditions and for a sufficient time periodto allow the bound population of product-secreting cells to grow and torelease a second-secreted product concentration into the culture medium;and harvesting product by removing from the bioreactor at least aportion of the culture medium with the second-secreted productconcentration from the bioreactor while leaving the scaffold with thebound population of product-secreting cells in the bioreactor.
 2. Themethod of claim 1, wherein the product is chosen from a virus, anantibody, a growth factor, a protein, a peptide, and a hormone.
 3. Themethod of claim 1, wherein the product is a protein and the protein isan interferon.
 4. The method of claim 1, wherein the product is a virusand the virus is optionally a flavivirus.
 5. A method of increasingvirus yield per culture in cells growing on a conditioned microcarrierin a bioreactor, the method comprising: transferring a plurality of seedcells into the bioreactor containing a microcarrier and an originalvolume of a culture medium, cultivating the seed cells to bind to themicrocarier and form a bound cell population that is from about 35percent to about 95 percent confluent on the microcarrier; removing fromthe bioreactor from about 30 percent to about 88 percent of the originalvolume of the culture medium in the bioreactor, while leaving themicrocarrier with the bound cell population in the bioreactor, to form afirst reduced volume of culture medium; infecting the bound cellpopulation with a virus, which can optionally be a flavivirus; addingfresh culture medium to the bioreactor to maintain the bound cellpopulation; culturing the bound cell population for a sufficient timeperiod to allow the infected, bound cell population to release a shedvirus concentration into the culture medium in the bioreactor; andharvesting virus by removing from the bioreactor at least a portion ofthe culture medium with shed virus therein.
 6. The method of claim 5,wherein the method further comprises: semi-harvesting virus by removingfrom the bioreactor a portion of the culture medium with a first-shedvirus concentration, the portion of the culture medium removed equal tofrom about 50 percent to about 90 percent of the original volume of theculture medium, while leaving the microcarrier with the infected, boundcell population, and a second reduced volume of the culture medium inthe bioreactor; re-feeding the bound cell population by adding to thebioreactor a second amount of a fresh culture medium sufficient toincrease the second reduced volume of the culture medium in thebioreactor to approximately the original volume of the culture medium;culturing the bound cell population in the bioreactor under sufficientconditions and for a sufficient time period to allow the virus tocontinue to allow the infected, bound cell population to release asecond-shed virus into the culture medium in the bioreactor; andharvesting virus by removing from the bioreactor at least a portion ofthe culture medium with the second-shed virus while leaving themicrocarrier with the infected, bound cell population in the bioreactor.7. The method of claim 6, wherein the semi-harvesting of virus comprisesremoving about 65 percent to about 75 percent of the culture medium withthe first-shed virus from the bioreactor.
 8. The method of claim 5,wherein the infecting of the bound cell population with a virus isperformed when the bound cell population is about 85 percent confluenton the microcarrier.
 9. The method of claim 1, wherein the bioreactor isa bench-top bioreactor, spinner flask or a disposable bioreactor havinga volume of from about 25 liters to about 200 liters.
 10. The method ofclaim 1, wherein the cells are anchorage-dependent mammalian cells andoptionally are VERO cells.
 11. A method of making a product from a cellculture of product-secreting cells bound to a scaffold, which canoptionally be a microcarrier, at least partially immersed in an originalvolume of a culture medium in a bioreactor, the method comprising:semi-harvesting product by removing from the bioreactor a first portionof the original volume of the culture medium with a first-secretedproduct concentration from the bioreactor while leaving the scaffoldwith the bound population of product-secreting cells in the bioreactor;re-feeding the bound population of product-secreting cells by adding tothe bioreactor an amount of a fresh culture medium sufficient toincrease the volume of the culture medium in the bioreactor toapproximately the original volume of the culture medium; agitating theculture medium in the bioreactor under sufficient conditions and for asufficient time period to allow the bound population ofproduct-secreting cells to grow and to release a second-secreted productconcentration into the culture medium; and harvesting product byremoving from the bioreactor at least a portion of the culture mediumwith the second-secreted product concentration from the bioreactor whileleaving the scaffold with the bound population of product-secretingcells in the bioreactor.
 12. The method of claim 11, wherein the productis chosen from a virus, an antibody, a growth factor, a protein, apeptide, and a hormone.
 13. The method of claim 11, wherein the productis a protein and the protein is an interferon.
 14. The method of claim11, wherein the product is a virus and the virus is optionally aflavivirus.
 15. A method of making a virus in cells growing on aconditioned microcarrier in a bioreactor, the method comprising:transferring a plurality of seed cells into the bioreactor containing amicrocarrier and an original volume of a culture medium, cultivating theseed cells to bind to the microcarier and form a bound cell populationthat is from about 35 percent to about 95 percent confluent on themicrocarrier; removing from the bioreactor from about 30 percent toabout 88 percent of the original volume of the culture medium in thebioreactor, while leaving the microcarrier with the bound cellpopulation in the bioreactor, to form a first reduced volume of culturemedium; infecting the bound cell population with a virus, which canoptionally be a flavivirus; adding fresh culture medium to thebioreactor to maintain the bound cell population; culturing the boundcell population for a sufficient time period to allow the infected,bound cell population to release a shed virus concentration into theculture medium in the bioreactor; and harvesting virus by removing fromthe bioreactor at least a portion of the culture medium with shed virustherein.
 16. The method of claim 15, wherein the method furthercomprises: semi-harvesting virus by removing from the bioreactor aportion of the culture medium with a first-shed virus concentration, theportion of the culture medium removed equal to from about 50 percent toabout 90 percent of the original volume of the culture medium, whileleaving the microcarrier with the infected, bound cell population, and asecond reduced volume of the culture medium in the bioreactor;re-feeding the bound cell population by adding to the bioreactor asecond amount of a fresh culture medium sufficient to increase thesecond reduced volume of the culture medium in the bioreactor toapproximately the original volume of the culture medium; culturing thebound cell population in the bioreactor under sufficient conditions andfor a sufficient time period to allow the virus to continue to allow theinfected, bound cell population to release a second-shed virus into theculture medium in the bioreactor; and harvesting virus by removing fromthe bioreactor at least a portion of the culture medium with thesecond-shed virus while leaving the microcarrier with the infected,bound cell population in the bioreactor.
 17. The method of claim 16,wherein the semi-harvesting of virus comprises removing about 65 percentto about 75 percent of the culture medium with the first-shed virus fromthe bioreactor.
 18. The method of claim 15, wherein the infecting of thebound cell population with a virus is performed when the bound cellpopulation is about 85 percent confluent on the microcarrier.
 19. Themethod of claim 1, wherein the bioreactor is a bench-top bioreactor,spinner flask or a disposable bioreactor having a volume of from about25 liters to about 200 liters.
 20. The method of claim 1, wherein thecells are anchorage-dependent mammalian cells and optionally are VEROcells.